Abstract
We describe a regulatable selective system which facilitates the isolation of high expression mammalian cells. The system is based on a modified tetracy-cline inducible expression system where the binding of the tetracycline-controlled rtTA transactivator to the tetracycline responsive promoter PhCMV*-1 depends on the presence of doxycycline (Dox). When the cells that producefunctional tetracycline-controlled transactivator rtTA and express the dominant selectable marker bsr gene under the control of the tetracycline responsive promoter PhCMV*-1 are cultured in the presence of sufficient amount of the inducer (1–2 μg/ml Dox), the rtTA transactivators bind to the PhCMV*-1 promoter and drive the transcription of the bsr gene. The cells therefore are resistant to the selective agent blasticidin S (5 μg/ml). However, when the cells are cultured in a medium containing the concentration of blasticidin S but only very low concentration of the inducer Dox (1 ng/ml or lower) to repress the rtTA-dependent transcription of the bsr gene, only the cells capable of expression of the bsr gene relatively independent of Dox can survive. These cells, when cultured in the presence of sufficient amount of inducer (1–2 μg/ml Dox), express much higher level of bsr gene than the parent population. By co-transfecting a target gene with the bsr gene using calcium phosphate co-precipitation, high expression cells can be isolated with the same selective procedures. As the regulatable selective system uses a dominant selectable marker, it can be used in prototrophic mammalian cells. Theoretically, the same strategy can be extended to other eukaryotic expression systems.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kaufman, R.J. (1990) in Goeddel, D.V. (ed.), Methods in Enzymology. Academic Press. San Diego, Vol. 185, pp. 537–566.
Gossen, M. et al. (1995). Science, 268, 1766–1769.
Izumi, M. et al. (1991) Exp. Cell Res., 197, 229–233.
Chen, C. and Okayama, H. (1987) Mol. Cell. Biol., 7, 2745–2752.
Baron, D., Freundieb, S., Gossen, M. and Bujard, H. (1995) Nucleic Acids Res., 23, 3605–3606.
Dirks, W., Wirth, M. and Hauser, H. (1993) Gene, 128, 247–249.
Totsuka, M. et al. (1990) Agric. Biol Chem., 54, 3111–3116.
Author information
Authors and Affiliations
Editor information
Rights and permissions
Copyright information
© 1999 Kluwer Academic Publishers
About this chapter
Cite this chapter
Teruya, K., Zhang, YP., Katakura, Y., Shirahata, S. (1999). A Regulatable Selective System Facilitating Isolation of High Expression Mammalian Cells. In: Bernard, A., Griffiths, B., Noé, W., Wurm, F. (eds) Animal Cell Technology: Products from Cells, Cells as Products. Springer, Dordrecht. https://doi.org/10.1007/0-306-46875-1_13
Download citation
DOI: https://doi.org/10.1007/0-306-46875-1_13
Publisher Name: Springer, Dordrecht
Print ISBN: 978-0-7923-6075-9
Online ISBN: 978-0-306-46875-9
eBook Packages: Springer Book Archive